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Genetic toxicity in vitro

Description of key information

In three bacterial reverse mutation assays a weak positive reaction was observed. In a mammalian cell gene mutation assay in CHO cells no mutagenic activity was observed.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Jan 2017 - 19 Jan 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
his, trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from induced rats
Test concentrations with justification for top dose:
0; 33; 100; 333; 1000; 2600 and 5200 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 37°C for 48 – 72 hours in the dark,

NUMBER OF REPLICATIONS: three

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants (factor ≤ 0.6); clearing or diminution of the background lawn (= reduced his- or trp- background growth)


POSITIVE CONTROLS

With S9 mix
• 2-aminoanthracene (2-AA)
- 2.5 μg/plate, dissolved in DMSO for strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO for strain: Escherichia coli WP2 uvrA

Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
- 5 μg/plate, dissolved in DMSO for strains: TA 1535, TA 100

• 4-nitro-o-phenylenediamine (NOPD)
- 10 μg/plate, dissolved in DMSO for strain: TA 98

• 9-aminoacridine (AAC)
- 100 μg/plate, dissolved in DMSO for strain: TA 1537

• 4-nitroquinoline-N-oxide (4-NQO)
- 5 μg/plate, dissolved in DMSO for strain: E. coli WP2 uvrA
Rationale for test conditions:
Mutagenicity was observed in the standard plate test. Therefore, the standard plate test was repeated instead of the prival modification.
Evaluation criteria:
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 333 μg/plate onward with and without S9 mix.

HISTORICAL CONTROL DATA
In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies within or nearby the range of the historical positive control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions from about 2600 μg/plate onward.
Conclusions:
Under the experimental conditions of this study, the test substance is mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA in a reverse mutation assay. The dose range applied was 33 μg - 5200 μg/plate in a standard plate test (SPT) with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from about 333 μg/plate onward with and without S9 mix. A bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2600 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test either with or without metabolic activation using tester strains TA 1535, TA 100 TA 1537 and E. coli WP2 uvrA. A relevant, reproducible and partly dose dependent increase in the number of his+ revertants exceeding a factor of 2 compared to the concurrent vehicle control was observed with TA 98 without and with metabolic activation.

TA 98 without S9 mix: Increase of revertants at concentrations of 1000, 2600 and 5200 μg/plate in the 1st and 2nd Experiment.

TA 98 with S9 mix: Increase of revertants at a concentration of 5200 μg/plate in the 1st and 2nd Experiment.

Under the experimental conditions of this study, the test substance is mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-04-15 to 1996-12-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham´s F10 medium supplemented with 10% fetal calf serum as well as 100 U/mL penicillin and 10 µg/mL streptomycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (Aroclor induced rat liver)
Test concentrations with justification for top dose:
Experiment 1:
(+) S9: 2.22, 6.66, 20, 60 µg/mL
(-) S9: 1.11, 3.33, 10, 30 µg/mL

Experiment 2:
(+) S9: 1.875, 3.75, 7.5, 15 µg/mL
(-) S9: 0.9375, 1.875, 3.75, 7.5 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours with S9 mix; 21 hours without S9 mix
- Expression time (cells in growth medium): 7 - 8 days at 37 °C
- Selection time: 7 - 8 days at 37 °C
- Fixation time (start of exposure up to fixation or harvest of cells): 14 - 16 days

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Cultures were treated in duplicate.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Acceptance criteria
The results of the experiments should not be influenced by a technical error, contamination or a recognized artifact. From each experiment, at least three concentrations of the test substance, one positive and one solvent control should be evaluated. The mutant frequency of the solvent controls (spontaneous mutant frequency) should not exceed 35x10^-6. The positive control should fulfil the criteria for a mutagenic substance. The highest concentration of the test substance applied in the mutagenicity test should either reduce the viable cells by about 50-90% or correspond to the test substance's solubility limit (precipitates in the culture). In case of non-toxic freely soluble compounds the highest tested concentration will be 5 mg/mL.

Criteria for a positive response
The test substance will be considered to be mutagenic if
• The assay is valid (see assay acceptance criteria)
• The mutant frequency at one or more concentrations is significantly greater than that of the negative control and the number of normalized mutant clones in the treated and untreated cultures differs by more than 20.
• There is a significant dose-relationship as indicated by the linear trend analysis.
• The effects described above are reproducible.


Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Arlett, C.F., DM. Smith, M.H.L. Green, D.B. McGregor, G.M. Clarke, J. Cole, JC. Asquith (1990); Mammalian cell gene mutation assays based upon colony formation. In: Statistical Evaluation of Mutagenicity Test Data (ed Kirkland, D.J.); Cambridge University press, pp 66-101)
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

RANGE-FINDING/CYTOTOXICITY STUDIES:
In the preliminary toxicity test with and without metabolic activation 12 concentrations of the test substance were tested. The concentrations selected ranged from 0.98 to 2000.00 µg/mL and separated by 2-fold intervals. In the part with metabolic activation the test material proved to be markedly toxic down to the concentration of 62.50 µg/mL. At the concentration of 31.25 µg/mL, the acute growth inhibition was still 70.3%, while the next lower concentration of 15.63 µg/mL was not toxic. In the part without metabolic activation the test substance exerted a strong growth inhibitory effect down to the concentration of 31.25 µg/mL. The next lower concentration revealed an acute inhibition of growth of 41.6%.

COMPARISON WITH HISTORICAL CONTROL DATA:
The comparison of the historical control data with the results of the positive and negative controls has not disclosed any inconsistencies.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1 Results

Experiment

Exposure period [h]

Dose (µg/mL)

S9 mix

Prec.

Mean mutant frequency
[x 10^-6]

1

21

Negative control

-

-

2.71

 

 

1.11

-

-

3.09

 

 

3.33

-

-

2.87

 

 

10

-

-

3.13

 

 

30

-

-

*

 

 

Positive control

-

-

1243.41

 

 

 

 

 

 

2

21

Negative control

-

-

3.15

 

 

0.9375

-

-

2.68

 

 

1.875

-

-

2.39

 

 

3.75

-

-

2.73

 

 

7.5

-

-

2.57

 

 

Positive control

-

-

1480.85

 

 

 

 

 

 

1

5

Negative control

+

-

3.01

 

 

2.22

+

-

2.94

 

 

6.66

+

-

2.57

 

 

20

+

-

4.41

 

 

60

+

-

*

 

 

Positive control

+

-

93.90

 

 

 

 

 

 

2

5

Negative control

+

-

2.91

 

 

1.875

+

-

1.92

 

 

3.75

+

-

2.30

 

 

7.5

+

-

2.14

 

 

15

+

-

2.70

 

 

Positive control

+

-

91.73

* No data

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay- Ames

The test substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli according to OECD guideline 471. The following strains were used: S. typhimurium TA 98, TA 100, TA 102, TA 1535, TA 1537 and E. coli WP2 uvrA. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was dissolved in DMSO and tested at five concentrations of 312.5, 625, 1250, 2500, 5000 µg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at the same concentrations. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable known mutagen as positive control. The original experiment with and without metabolic activation and the confirmatory experiment without activation were performed as standard plate incorporation assay. The confirmatory experiments with metabolic activation were carried out as preincubation assay. In the experiments performed without metabolic activation, treatment of strains TA 1537 and TA 98 with the test substance led to a very weak concentration-dependent increase in the number of histidine-prototrophic back-mutants at the highest concentration each. A similar effect was also observed in the confirmatory experiment with activation performed on strain 1537. It is assumed that the source of the mutagenic action is a contaminant rather than the main component of the test substance.

In a second Ames test, the test substance was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium equivalent to OECD guideline 471. The investigations were performed on strains TA 98, TA 100 and TA 1537 with the following concentrations of the test substance without and with microsomal activation: 20, 78, 313, 1250 and 5000 µg/0.1 mL. These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substance formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors). In the experiment performed without microsomal activation, treatment with the test substance led to a slight increase in the number of back-mutant colonies of the strains TA 98 and TA 1537 at the highest concentration by comparison with the negative control. But precipitation of the test substance was observed in the two highest test concentrations. In the experiment carried out with microsomal activation, none of the tested concentrations of the test substance led to an increase in the incidence of histidine-prototrophic mutants by comparison with the control. Thus, the test substance treatment led to a very weak concentration-dependent increase in the number of back mutants at the highest concentration each.

In the most recent Ames study, the test substance was tested for its mutagenic potential based on the ability to induce point mutations in bacterial strains Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA in a reverse mutation assay. Test substance precipitation was found from about 333 μg/plate onward with and without S9 mix. A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions from about 2600 μg/plate onward. According to the results of this study the test substance led to a relevant and partly dose dependent increase in the number of his+ revertants with the tester strain TA 98 with and without S9 mix. The increase of revertants was reproducible in two experiments carried out independently of each other. Based on the recent assessment criteria the test substance has to be considered positive. The results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.

In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies within or nearby the range of the historical positive control data. In conclusion, under the experimental conditions chosen here, it is concluded that the test item is a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Mammalian cell gene mutation test

The test substance was tested for mutagenic effects on V79 Chinese hamster cells in vitro according to OECD guideline 476. The test substance was dissolved in DMSO. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours. The results of each experiment were confirmed in a second and independent experiment (confirmatory experiment). The experiment with metabolic activation was performed at the following concentrations: 2.22, 6.67, 20.00 and 60.00 µg/mL. At the highest concentration, no cells at all were present, due to toxicity. The mean growth inhibiting values found at the next lower concentration after treatment and expression were 99.2 % and 33.9 % respectively. In the confirmatory experiment the concentrations applied were 1.88, 3.75, 7.50 and 15.00 µg/mL. The highest concentration revealed a mean acute growth inhibition of 72.4 %. The mean growth inhibitory effect after the expression period was 10.3 %. N-Nitrosodimethylamine (DMN, 1.0 µL/mL) was used as positive control. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG). The experiment without metabolic activation was performed at the following concentrations: 1.11, 3.33, 10.00 and 30.00 µg/mL. The highest concentration of 30.00 µg/mL resulted in a cytotoxicity of 99.8 % and was completely toxic both at subculture and after expression. The mean growth inhibition values found at the next lower concentration after treatment and expression were 98.5 % and 10.6 %, respectively. In the confirmatory experiment the concentrations applied were 0.94, 1.88, 3.75 and 7.50 µg/mL. The highest concentration revealed a mean cytotoxicity of 80.6 %. The mean growth inhibition after the expression period was 12.6 %. Ethylmethansulfonate (EMS, 0.3 µL/mL) was used as positive control. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG. Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that the test substance and its metabolites did not show any mutagenic activity in this forward mutation system.

Conclusion

The test substance showed a weak positive response in three different bacterial reverse mutation assays in the strains TA98 and TA1537 without metabolic activation. The mutation rate was increased at the highest tested concentration. This weak mutagenic response could not be verified in mammalian cell culture. A mammalian cell gene mutation test in Chinese hamster ovary cells did not show an increase of the mutant frequencies at any of the tested concentrations with or without metabolic activation.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance does not need to be classified and labelled as mutagenic under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation (EC) No 605/2014.